The primary objectives of this project are to elucidate how replication- dependent and replication-independent histone synthesis are interrelated and regulated with regard to the stoichiometry of the various histone subtypes in the nucleosome, and how replication-dependent histone synthesis is regulated with regard to the rate of DNA synthesis. Because of exciting finds in the study of the H2A.X gene, most of our effort this year has been applied to that study. (see Z01 CM 06192-03). We have isolated some H2A cDNA clones which suggest that various specific mechanisms permit histone synthesis in quiescent cells to be from the same genes as used in proliferating cells. We are in the process of characterizing these mechanisms. We have evidence suggesting that soluble histone in mammalian cells is bound in a negatively charged complex that sediments at 8-9s. Because histones have been so conserved during evolution, it has not been possible to prepare high affinity antibodies. We are currently testing the possibility of preparing transfected cell lines carrying genes for altered histone proteins that carry an epitope to a known monoclonal antibody. Such an approach would greatly increase our ability to isolate and characterize components bound to the soluble histone complex. Isolating and characterizing the components involved in the complex but rigorous coordination of histone and DNA synthesis may lead to techniques or compounds of therapeutic value which disrupt this coordination in such a manner as to be selectively lethal to proliferating cells.